Multiple growth factor compositions, methods of fabrication, and methods of treatment

ABSTRACT

Disclosed are compositions with sustained-release carriers associated with at least two different types of growth factors and methods of fabrication and treatments thereof. In some embodiments, simultaneous release of the growth factors may be preferred while in other embodiments, sequential release of the growth factors may be preferred. Application of at least two growth factors to an injury site, e.g., compromised cardiac tissue caused by, for example, myocardial infarction or ischemic heart failure, may better mimic and induce the complex growth factor signaling pathways necessary to improve cardiac function. When applied to a patient after a myocardial infarction or ischemic heart failure, multiple growth factors within a sustained-release carrier platform or platforms may cause a synergistic effect on injected cells intending to alleviate left ventricle remodeling. Methods of treatment include percutaneous, sub-xiphoid, and open chest methods using catheters and/or syringes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of co-pending application Ser. No.11/877,635, filed Oct. 23, 2007.

FIELD OF THE INVENTION

Post-myocardial infarction compositions and methods of treatment.

BACKGROUND OF THE INVENTION

Ischemic heart disease typically results from an imbalance between themyocardial blood flow and the metabolic demand of the myocardium.Progressive atherosclerosis with increasing occlusion of coronaryarteries leads to a reduction in coronary blood flow. “Atherosclerosis”is a type of arteriosclerosis in which cells including smooth musclecells and macrophages, fatty substances, cholesterol, cellular wasteproduct, calcium and fibrin build up in the inner lining of a bodyvessel. “Arteriosclerosis” refers to the thickening and hardening ofarteries. Blood flow can be further decreased by additional events suchas changes in circulation that lead to hypoperfusion, vasospasm orthrombosis.

Myocardial infarction (MI) is one form of heart disease that oftenresults from the sudden lack of supply of oxygen and other nutrients.The lack of blood supply is a result of a closure of the coronary artery(or any other artery feeding the heart) which nourishes a particularpart of the heart muscle. The cause of this event is generallyattributed to arteriosclerosis in coronary vessels.

Formerly, it was believed that an MI was caused from a slow progressionof closure from, for example, 95% then to 100%. However, an MI can alsobe a result of minor blockages where, for example, there is a rupture ofthe cholesterol plaque resulting in blood clotting within the artery.Thus, the flow of blood is blocked and downstream cellular damageoccurs. This damage can cause irregular rhythms that can be fatal, eventhough the remaining muscle is strong enough to pump a sufficient amountof blood. As a result of this insult to the heart tissue, scar tissuetends to naturally form.

An important component in the progression to heart failure is remodelingof the heart due to mismatched mechanical forces between the infarctedregion and the healthy tissue resulting in uneven stress and straindistribution in the left ventricle (LV). Once an MI occurs, remodelingof the heart begins. The principle components of the remodeling eventinclude myocyte death, edema and inflammation, followed by fibroblastinfiltration and collagen deposition, and finally scar formation. Theprinciple component of the scar is collagen. Since mature myocytes of anadult are not regenerated, the infarct region experiences significantthinning. Myocyte loss is the major etiologic factor of wall thinningand chamber dilation that may ultimately lead to progression of cardiacmyopathy. In other areas, remote regions experience hypertrophy(thickening) resulting in an overall enlargement of the left ventricle.This is the end result of the remodeling cascade. These changes in theheart result in changes in the patient's lifestyle and their ability towalk and to exercise. These changes also correlate with physiologicalchanges that result in increase in blood pressure and worsening systolicand diastolic performance.

Currently, methods to alleviate LV remodeling, including application ofcells, biomaterials (also known as “bioscaffoldings”), or cell-loadedbioscaffoldings to an injury site (e.g., compromised heart tissue), havebeen preliminarily explored. Implantation of autologous cells fordamaged myocardium is under current clinical investigation. In otherrecent studies, implanting biomaterials to an infarct region has beenshown to improve the ejection fraction in rats. See Christman, K. L.,Biomaterials for the Treatment of Myocardial Infarction, J. AmericanCollege of Cardiology, vol. 48, no. 5 (2006). Limitations of currentmethods include low cell retention at the injury site and reducedlong-term viability of injected or endogenous cells.

Growth factors are naturally occurring proteins secreted by manydifferent cell types for signaling to induce cell migration,differentiation, survival, or proliferation, in addition to otherfunctions. Signaling occurs through binding of factors to cell surfacespecific receptors. Signals can be amplified within the cell to regulatespecific gene expression. Growth factors typically act in a dose- andtime-dependent fashion with small variations in concentrations resultingin a biological effect. When applied in post-MI therapies, growthfactors have the potential to increase the survival of cells whetherendogenous or exogenous. Current growth factor therapies for both acuteMI and HF have focused on bolus or systemic injection of a single growthfactor type. Such therapies, however, are subject to a large percentageof the growth factor being washed away by blood flow thus minimizing thepotential benefit of the treatment agent that may otherwise be obtained.Moreover, application of a single growth factor may not be as beneficialas previously hypothesized in view of naturally occurring complex growthfactor signaling pathways.

SUMMARY OF THE INVENTION

A composition including a first bioerodable carrier platform capable ofsustained release of a treatment agent, the composition including atleast two different treatment agents, at least one of the two differenttreatment agents associated with the first bioerodable carrier platformwherein each treatment agent has a function, when delivered tocompromised heart tissue, selected from the group consisting of cellsurvival, cell recruitment, angiogenesis, arteriogenesis, andanti-fibrotic development, and wherein the at least two differenttreatment agents have a different function relative to one another. Thecomposition may further include at least one second differentbioerodable carrier platform capable of sustained release of a treatmentagent wherein a rate of release of the first bioerodable carrierplatform is different relative to a rate of release of the secondbioerodable carrier platform, and wherein one of the treatment agents isassociated with the first bioerodable carrier platform and the other ofthe treatment agents is associated with the second different bioerodablecarrier platform.

A method of treating compromised heart tissue within a mammal, includingthe process of advancing a delivery device through a lumen of a bloodvessel to a treatment region wherein the treatment region is compromisedheart tissue; and introducing a composition through the delivery devicewherein the composition includes (i) a first bioerodable carrierplatform capable of sustained release of a treatment agent; and (ii) atleast two different treatment agents, at least one of the two differenttreatment agents associated with the first bioerodable carrier platformwherein the treatment agents have a function, when delivered tocompromised heart tissue, selected from the group consisting of cellsurvival, cell recruitment, angiogenesis, arteriogenesis, andanti-fibrotic development, and wherein the at least two differenttreatment agents have a different function relative to one another.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate an alternative embodiment of a dual-needleinjection device which may be used to deliver core-shell particles inaccordance with embodiments of the invention.

FIG. 2 illustrates an embodiment of a syringe which may be used pursuantto embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention include compositions comprised ofsustained-release carriers associated with at least two different typesof growth factors. In some embodiments, simultaneous release of thegrowth factors may be preferred while in other embodiments, sequentialrelease of the growth factors may be preferred. It is anticipated thatthe application of at least two growth factors to an injury site, e.g.,compromised cardiac tissue caused by, for example, myocardial infarctionor ischemic heart failure, may better mimic and induce the complexgrowth factor signaling pathways necessary to improve cardiac function.In vitro results have demonstrated that multiple growth factors cause asynergistic effect on cultured cells, i.e., the effect of two factors isgreater than the sum of their two-fold effect.

Carriers

According to embodiments of the invention, a bioerodable carrierplatform associated with at least two treatment agents can be used forthe sustained or controlled release of the treatment agents to achieve asynergistic effect to treat or prevent the formation of compromisedcardiac tissue. In the context of this application, “associated with”means encapsulated, suspended, disposed within or on (chemisorbed) thecarrier platform. The treatment agent(s) can be, for example, at leasttwo different growth factors. It is anticipated that a sustained-releasebioerodable carrier platform (or multiple sustained-release carrierplatforms) associated with at least two growth factors may be beneficialby mimicking and inducing the complex growth factor signaling pathwaysnecessary to improve cardiac function when applied to a post-MI infarctor HF region. Sustained-release carriers may include, but are notlimited to: particles, such as microparticles, nanoparticles, orcore-shell particles; fibers, such as, microfibers or nanofibers;vesicles, such as liposomes, polymerosomes, micelles, or microbubbles;or hydrogels (also known as “bioscaffoldings” when administered tocompromised cardiac tissue) which may be formulated as a singlecomponent or a multiple component systems.

In one embodiment, the biodegradable carrier platform is a liposome.“Liposomes” are artificial vesicles that are approximately spherical inshape and can be produced from natural phospholipids, sphingolipids,ceramides, cholesterol or estradiol. Generally, a liposome has a lipidbilayer membrane encapsulating an aqueous solution, i.e., “core.” Thelipid bilayer membrane allows for fusion with an endogenous (orexogenous) cell membrane, which, similar to the liposome, comprises asemipermeable lipid bilayer. In one method, phospholipids and atreatment agent are mixed with estradiol in chloroform. Suitablephospholipids include, but are not limited to,dimyristoylphosphatidylcholine (DMPC), dipalmitoyl phosphatidylethanolamine (DPPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-dalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), eggphosphatidylcholine (EPC), hydrogenated egg phosphatidylcholine (HEPC),soybean phosphatidylcholine (SPC), hydrogenated soybeanphosphatidylcholine (HSPC). The liposomes may also be hydrophilicallymodified by coating the liposomes with an agent such as poly(ethyleneglycol) or dextran. Such coating tends to avoid detection from thebody's immune system. After mixing, the solvent (and an optionalco-solvent) can be evaporated with heat or ambient temperature in around bottom flask. Resultant lipids will be deposited on the glasssurface. The deposited lipid film can be re-suspended in aqueoussolution to form multilamellar (or unilamellar) vesicles, and extrudedto prepare appropriate sized liposomes. Liposomes can be in a range fromabout 25 nm to about 2000 nm. One of ordinary skill in the art willappreciate that the treatment agents to reside within the core of theliposome are likely substantially hydrophilic, as the core of theliposome is generally an aqueous environment.

In another embodiment, the biodegradable carrier platform is apolymerosome. “Polymerosomes” are polymer vesicles formed from di-blockor tri-block copolymers with blocks of differing solubility. Diblockcopolymers are known to spontaneously organize into polymer vesicles.Polymerosomes may be formed by methods such as film rehydration,electro-formation and double emulsion. In some methods, a similarmanufacturing technique can be used as that of a liposome to formpolymerosomes. In some embodiments, a polymerosome can be a di-blockcopolymer including a block which is hydrophobic, e.g., poly(lacticacid), polycaprolactone, n-butyl acrylate, and another block which ishydrophilic, e.g., poly(ethylene glycol), poly(acrylic acid). Apolymerosome can be in a range from between about 25 nm to about 2000nm. One of ordinary skill in the art will appreciate that thehydrophobic and hydrophilic regions of the treatment agents willgenerally associate with the hydrophobic and hydrophilic regions of thepolymerosome, respectively.

In another embodiment, the biodegradable carrier platform is a micelle.A “micelle” is an aggregate of surfactant or polymer molecules dispersedin a liquid colloid. Micelles are often globular in shape, but othershapes are possible, including ellipsoids, cylinders, bilayers, andvesicles. The shape of a micelle is controlled largely by the moleculargeometry of its surfactant or polymer molecules, but micelle shape alsodepends on conditions such as temperature or pH, and the type andconcentration of any added salt.

Micelles can be formed from individual block copolymer molecules, eachof which contains a hydrophobic block and a hydrophilic block. Theamphiphilic nature of the block copolymers enables them to self-assembleto form nanosized aggregates of various morphologies in aqueous solutionsuch that the hydrophobic blocks form the core of the micelle, which issurrounded by the hydrophilic blocks, which form the outer shell. Theinner core of the micelle creates a hydrophobic microenvironment formimetic peptide, while the hydrophilic shell provides a stabilizinginterface between the micelle core and an aqueous medium. Examples ofpolymers which can be used to form micelles include, but are not limitedto, polycaprolactone polyethylene oxide blocks, polyethyleneoxide-β-polypropylene oxide-β-polyethylene oxide triblock copolymer andcopolymers which have a polypeptide or polylactic acid core-formingblock and a polyethylene oxide block. A micelle can be in a range frombetween about 10 nm to about 100 nm. One of ordinary skill in the artwill appreciate that the hydrophobic and hydrophilic regions of thetreatment agent will generally associate with the hydrophobic andhydrophilic regions of the micelle, respectively.

In another embodiment, the biodegradable carrier platform is a particle.Various methods can be employed to formulate and infuse or load theparticles with growth factors. Representative methods include, but arenot limited to, water/oil/water or water/oil or water/water emulsionfollowed by solvent evaporation, electrohydrodynamic atomization (orelectrospraying), piezo-assisted spraying, air-assisted jetting, supercritical CO₂ and spray drying. In one example, the particles areprepared by a water/oil/water (W₁/O/W₂) double emulsion method. In theW₁ phase, a first aqueous phase is dispersed into the oil phaseconsisting of polymer (or other platform) dissolved in organic solvent(e.g., dichloromethane) and the growth factor(s) using a high-speedhomogenizer. Examples of polymers include, but are not limited to,poly(L-lactide-co-glycolide) (PLGA), poly(D,L-lactide-co-glycolide),poly(L-lactide), poly(D,L-lactide) (PLA), poly(ε-caprolactone),poly(L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone),cross-linked poly(ethylene glycol) (PEG), PLA-PEG co-polymers,poly-ester-amide co-polymers (PEA), and polyphosphazines. The primarywater-in-oil (W/O) emulsion is then dispersed in a second aqueoussolution containing a polymeric surfactant, e.g., poly(vinyl alcohol)(PVA) or PEG, and further homogenized to produce a W/O/W emulsion. Afterstirring for several hours, the particles are collected bycentrifugation or filtration. A microparticle can be in a range fromabout 1 μm to about 200 μm, preferably 5 μm to 15 μm. A nanoparticle canbe in a range from between about 10 nm to about 1000 nm, preferablyabout 250 nm to about 750 nm. Particles may also be fabricated from, forexample, dextran, alginate, cellulose, hyaluronan, chitosan, collagen,albumin, gelatin, or like materials.

In a preferred embodiment, the biodegradable carrier platform is acore-shell particle. In the context of this application, “core-shell”means that the particle has a shell and includes from one to severalcore(s). Core-shell particles can be formed using various techniquessuch as, for example, coaxial electrospraying. In one exemplary methodof fabricating core-shell particles, a first liquid solution (L₁) may besupplied to an outer tube by a pump and a second different liquidsolution (L₂) may be supplied to an inner tube by a pump to form thecore-shell particles. Solution L₁ may be the precursor solution thatforms the (hydrophobic or hydrophilic) “shell” while solution L₂ may bethe precursor solution that forms the (hydrophilic or hydrophobic)“core” of the particles that will be eventually collected on acollection target as the electrospray system is being operated. Bycreating core-shell particles in which the “shell” and the “core”independently harbor different growth factors, different releaseprofiles may be obtained as the core and shell independently erode afterdelivery to a treatment site over a period of time (conditiondependent).

In one embodiment, a core-shell particle includes a shell, a first core,and a second core. A solution for the shell includes a hydrophobicpolymer in a molecular weight range of between 200 Daltons and 500,000Daltons and a concentration of between 0.01 mg/mL and 1000 mg/mL (weightpercent), e.g., poly(lactide-co-glycolide) or poly(ε-caprolactone). Afirst solution used to fabricate the first core includes a hydrophilicpolymer in a molecular weight range of between 200 Daltons and 1,500,000Daltons and a concentration of between 0.01 mg/mL and 1000 mg/mL (weightpercent), e.g., poly(ethylene glycol) or polyvinyl alcohol. Incorporatedwithin the first solution is a growth factor, such as vascularendothelial growth factor at a concentration of between 0.010 μg/mL toabout 10,000 μg/mL. A second solution used to fabricate the second corealso includes a hydrophilic polymer in a molecular weight range ofbetween 200 Daltons and 1,500,000 Daltons and a concentration of between0.01 mg/mL and 1000 mg/mL (weight percent), e.g., poly(ethylene glycol)or polyvinyl alcohol. Incorporated within the second solution is agrowth factor, such as hepatocyte growth factor at a concentration ofbetween 0.010 μg/mL to about 10,000 μg/mL. The particle may befabricated such that the release of vascular endothelial growth factorand hepatocyte growth factor are simultaneously or sequentiallyreleased. Release will depend on the chemical and/or physical nature ofthe platform (e.g., polymer) used as well as parameters such as, but notlimited to, molecular weight, concentration, and the addition or absenceof excipients within the solution(s).

In some embodiments, the biodegradable carrier platform is a microfiberor nanofiber. For example, the treatment agent-infused microfiber can beformulated by electrospinning “Electrospinning” is a process by whichmicrofibers are formed by using an electric field to draw a polymersolution from the tip of a capillary to a collector. A voltage isapplied to the polymer solution which causes a stream of solution to bedrawn toward a grounded collector. Electrospinning generates a web offibers which can be subsequently processed into smaller lengths.

Examples of sustained-release polymers which can be used inelectrospinning include, but are not limited to, PLGA copolymers, PLA,poly(ε-caprolactone) or PLA-PCL co-polymers, PEA, PEG copolymers,polyurethanes, polyurethane ureas, polyphosphazines and collagen. In onemethod, the treatment agent is mixed with a bioerodable polymersolution, a solvent and a surfactant. Examples of surfactants caninclude, but are not limited to, anionic or cationic surfactants. Usefulanionic surfactants include, but are not intended to be limited to,bis(2-ethylhexyl) sodium sulfosuccinate (AOT), bis (2-ethylhexyl)phosphate (NaDEHP), tauroglycocholate, and sodium lauryl sulfate. Auseful cationic surfactant is tetradecyltrimethyl-ammonium bromide(TTAB). Other surfactants include polyvinyl alcohol, polysorbates (e.g.,TWEEN® types) and poloxamers (e.g., PLURONIC® types). Examples of asolvent include, but are not limited to, hexafluoroisopropanol, acetone,or tetrahydrofuran/dimethyl acetamide blends. The treatmentagent-infused polymer solution is then subjected to electrospinning. Asthe solvent evaporates during electrospinning, the treatment agentincorporates and distributes within the polymer by non-covalentinteractions. The resultant microfibers which can be from about 0.05 μmto about 20 μm in diameter form a web which may then be subsequentprocessed into smaller lengths of about 5 μm to about 500 μm.

In one embodiment, fibers can be processed or electrospun from acollagen and elastin solution in hexafluoroisopropanol (HFP). Atreatment agent (e.g., growth factor) can be added to the biopolymersolution. A surfactant and a stabilizer can be used to evenly dispersethe treatment agent in the solvent. The polymer solution can then beloaded into a syringe and placed in a syringe pump for metereddispensing at a predetermined rate. A positive output lead of a highvoltage supply can be attached to a needle on the syringe. The needlecan be directed to a stainless steel grounded target placedapproximately 5-20 cm from the needle tip, which can be rotated at apredetermined speed to ensure an even coating. The distance of theneedle from the target can be varied depending upon the diameter of thefibers needed. The resultant microfibers are from about 0.05 μm to about20 μm in diameter and the resulting non-woven mat of fibers can then beprocessed into smaller lengths of about 5 μm to about 500 μm.

In another embodiment, the bioerodable carrier platform is a hydrogel,i.e., a bioscaffolding. A bioscaffolding may be a single component ormulti-component hydrogel fabricated from a single precursor or multipleprecursors. In one embodiment, a bioscaffolding formed of multipleprecursors may include two precursors which are in a liquid state beforemixing, and then a semi-solid gel-like state upon co-mixing thereof by amechanism such as, for example, physical or chemical cross-linkingExamples of hydrogels include, but are not limited to, hyaluronic acidor a salt thereof, fibrin glue, alginate, PEG copolymers, andsilk-elastin copolymers. One of ordinary skill in the art willappreciate that the treatment agents may be dispersed throughout, andtherefore associated with, any of the single or multi-componentprecursor solution(s).

In a still further embodiment, the bioerodable carrier platform is alipid-coated microbubble (LCM) including the peptide. Peptides can beincorporated into the microbubbles in a number of different ways,including binding of a peptide to the microbubble shell and attachmentof site-specific ligands. Perfluorocarbon-filled albumin microbubblesavidly bind proteins and synthetic peptides and are sufficiently stablefor circulating in the vasculature as blood pool agents. Thesemicrobubbles act as carriers of these agents until a site of interest isreached. Ultrasound applied over the skin surface can then be used toburst the microbubbles at a treatment site, causing localized release ofthe peptide or protein. Albumin-encapsulated microbubbles have alsodemonstrated a property to adhere to a vessel wall. These microbubblesprovide targeted delivery without the application of ultrasound.Microbubbles have also been shown to directly take up genetic material,such as plasmids and adenovirus, and phospholipid-coated microbubbleshave a high affinity for certain drugs.

The mechanisms by which ultrasound facilitates the delivery of drugs andgenes result from an interplay among the therapeutic agent, themicrobubble characteristics, the target tissue, and the nature ofultrasound energy. The presence of microbubbles in the insonified fieldreduces the peak negative pressure needed to enhance delivery withultrasound. This occurs because the microbubbles act as nuclei forcavitation, decreasing the threshold of ultrasound energy necessary tocause this phenomenon. The results of optical and acoustical studieshave suggested the following mechanisms for microbubble destruction byultrasound: gradual diffusion of gas at low acoustic power; formation ofa shell defect with diffusion of gas; immediate expulsion of themicrobubble shell at high acoustic power; and dispersion of themicrobubble into several smaller bubbles.

In general, polymers and biomacromolecules suitable to fabricate thebioerodable carrier platform(s) include, but are not limited to, fibringlue precursors, alginate gel precursors, combinations of fibrin glueand alginate glue precursors, small intestinal submucosa/urinary bladderde-cellularized matrix, synthetic poly(ethylene glycol)-based materials,poly(N-isopropylacrylamide) copolymers, elastomers such aspolyurethanes, polyurethane-ureas, and cross-linkable poly(ester)copolymers, in addition to dextran, silk-elastin, and hyaluronate.

In some embodiments, polymers used in the fabrication of the bioerodablecarrier platform may be surface-modified to attach ligands such asarginine-aspartate-glycine (RGD) to increase cell adhesion, spreadingand influence differentiation of a particular cell into the appropriatecell phenotype. For example, polymer particles or fibers may be treatedby radio frequency glow discharge under ammonia atmosphere to introduceamine groups onto the particle/fiber surface, which may be used toattach linear spacers (e.g., 1,4-diisocyanatobutane), which may in turnbe used to attach RGD groups.

Treatment Agents

According to some embodiments, a bioerodable carrier platform mayinclude at least two different treatment agents, such as growth factors.Growth factors participate in biochemical signaling pathways to inducecell migration, differentiation, survival, or proliferation. Signalingoccurs through binding of factors to cell surface specific receptors.Signals can be amplified within the cell to regulate specific geneexpression. Growth factors typically act in a dose- and time-dependentfashion with small variations in concentrations resulting in abiological effect. When applied in post-MI therapies, growth factorshave the potential to increase the survival of cells whether the cellsare endogenous or exogenous. It is anticipated that the application ofat least two growth factors to an injury site, e.g., compromised cardiactissue caused by, for example, myocardial infarction or ischemic heartfailure, may better mimic and induce the complex growth factor signalingpathways necessary to improve cardiac function. In vitro results havedemonstrated that multiple growth factors cause a synergistic effect oncultured cells, i.e., the effect of two factors is greater than the sumof their two-fold effect.

Examples of growth factors include, but are not limited to, vascularendothelial cell growth factor (VEGF), basic fibroblast growth factor(bFGF), platelet-derived growth factor (PDGF), platelet-derivedendothelial growth factor (PDEGF), insulin-like growth factor-1 (IGF-1),insulin-like growth factor-2 (IGF-2), transforming growth factor-alpha(TGF-α), transforming growth factor-beta (TGF-β), hepatocyte growthfactor (HGF), stem cell factor, stromal derived growth factor-1-alpha(SDF-1α), hematopoietic growth factor or granulocyte colony-stimulatingfactors (G-CSF), granulocyte macrophage colony-stimulating factors(GM-CSF), nerve growth factor (NGF), growth differentiation factor-9(GDF9), epidermal growth factor (EGF), neurotrophins, erythropoietin(EPO), thrombopoieten (TPO), myostatin (GDF-8), leukemia inhibitoryfactor (LIF), tumor necrosis factor-alpha (TNF-α), sonic hedgehog (Shh)protein (an upstream growth factor regulator).

In one embodiment, at least two growth factors can be associated with abioerodable carrier platform to target a particular response (or“effect” or “function”) useful for treating compromised cardiac tissue,such as a post-MI or HF region. Specific growth factors can produce aspecific response from specific cells or tissue types. The response mayinclude one of cell survival, angiogenesis, cell recruitment (orhoming), anti-fibrotic development, and/or extracellular matrixproduction.

Endogenous cardiomyocyte (myocytes) apoptosis is the major etiologicalfactor of wall thinning and chamber dilation and may ultimately lead toprogression of cardiac myopathy. After an infarction, mature myocytes ofan adult are not regenerated which can lead to significant thinning inthe infarct region. Thus, factors which promote cell survival and cellrecruitment applied to the infarct region are believed to be beneficial.

Angiogenesis, which is the promotion or causation of the formation ofnew blood vessels, is useful for treatment of a post-MI region for anumber of reasons. After an MI, the infarct tissue as well as the borderzone and the remote zone around the infarct tissue begin to remodel.Scar tissue forms in the infarct region as the granulation is replacedwith collagen. Stress from blood pressure cause the scar to thin out andstretch. The perfusion in this region is typically 10% of the healthyzone, decreasing the number of active capillaries. Increasing the numberof capillaries may lead to an increase in compliance of the ventricledue to filling up with blood. Other benefits of increasing blood flow tothe infarcted region include providing a route for circulating stemcells to seed and proliferate in the infarct region. Angiogenesis mayalso lead to increased oxygenation for the surviving cellular isletswithin the infarct region, or to prime the infarct region for subsequentcell transplantation for myocardial regeneration. In the border zone,surviving cells would also benefit from an increase in blood supplythrough an angiogenesis process. In the remote zone, where cardiac cellstend to hypertrophy and become surrounded with some interstitialfibrosis, the ability of cells to receive oxygen and therefore functionto full capacity are also compromised; thus, angiogenesis would bebeneficial in these regions as well.

Fibrosis is the formation or development of excess fibrous connectivetissue in an organ or tissue as a reparative or reactive process, asopposed to a formation of fibrous tissue as a normal constituent of anorgan or tissue. Excess formation of fibrous connective tissue on theheart may force the heart to work harder following a post-MI incident,thus impairing the restorative process. Thus, factors which decreaseproduction of excess fibrous connective tissue post-MI and applied tothe infarct region are believed to be beneficial.

The myocardial extracellular matrix (ECM) consists of macromolecules,primarily produced locally from fibroblasts, and includes a fibrillarcollagen network, a basement membrane and proteoglycans. The ECM of theheart was once believed to be an inert scaffold for cardiomyocytes butis now known to play an important role in LV remodeling. Excessproduction of fibrillar collagen in the ECM post-MI can cause myocardialstiffness which in turn causes the heart to have to work harder. As aresult, factors which influence the ECM and its respective constituentsapplied to the infarct region are believed to be beneficial.

In one embodiment, simultaneous release of two growth factors may beused to boost a single desired growth factor action or to productmulti-potent effects at an injury site such as a post-MI region. In oneexample, cell survival may be enhanced by the simultaneous release of(i) insulin-like growth factor-1 and hepatocyte growth factor, or (ii)insulin-like growth factor 1 and stem cell factor from a bioerodablecarrier platform. In another example, angiogenesis may be enhanced bysimultaneous release of (i) vascular endothelial growth factor andplatelet-derived growth factor, (ii) vascular endothelial growth factorand hepatocyte growth factor, (iii) vascular endothelial growth factorand stem cell factor, (iv) basic-fibroblast growth factor and hepatocytegrowth factor, (v) basic-fibroblast growth factor and stem cell factor,(vi) insulin-like growth factor 1 and basic-fibroblast growth factor;(vii) granulocyte colony-stimulating factor and hepatocyte growth factorfrom a bioerodable carrier platform. In another example, cellrecruitment may be enhanced by simultaneous release of (i) granulocytecolony-stimulating factor and hepatocyte growth factor, (ii) granulocytecolony-stimulating factor and stromal derived factor-1-alpha, (iii)granulocyte colony-stimulating factor and stem cell factor, or (iv)granulocyte colony-stimulating factor and insulin-like growth factor 1from a bioerodable carrier platform. Any of the above examples may bereferred to as a binary simultaneous release formulation. In someembodiments, the binary simultaneous release formulation may includesonic hedgehog protein (shh).

For simultaneous release formulations, binary or multiple growth factorsmay be encapsulated in a suitable bioerodable carrier platform(s), suchas liposomes, polymerosomes, micelles, particles (e.g., microparticles,nanoparticles, core-shell particles), nanofibers, or hydrogels.Simultaneous release formulations may be designed to produce similarrelease profiles over similar time spans of therapeutic doses of thegrowth factors. When delivered to an injury site (e.g., post-MI infarctsite or ischemic/chronic HF site) of a patient, simultaneous delivery ofthe growth factors may be achieved by fabricating a formulation whichencapsulates different growth factors within similar or the sameplatform. For example to achieve simultaneous release, at least twodifferent growth factors may be encapsulated within the same particleor, alternatively, within two different particles of the same orsubstantially the same composition, size, and porosity. In someembodiments, simultaneous release of the at least two treatment agentsmay be in a time interval of between ten seconds and 8 weeks or more.

In another embodiment, sequential release of two growth factors may beused to boost a single desired growth factor action or to productmulti-potent effects at an injury site (e.g., post-MI infarct site orischemic/chronic HF site). Sequential release may be beneficial in thecase when the second factor's effect is dependent on the action of thefirst growth factor, or, alternatively, warranted at a later point intime. In one example, cell survival followed by cell homing may beenhanced by the sequential release of (i) insulin-like growth factor 1followed by stromal derived factor-1-alpha, (ii) insulin-like growthfactor 1 followed by stem cell factor, (iii) insulin-like growth factor1 followed by hepatocyte growth factor, or (iv) insulin-like growthfactor 1 followed by granulocyte colony-stimulating factor from abioerodable carrier platform. In another example, anti-fibroboticdevelopment followed by angiogenesis may be enhanced by sequentialrelease of (i) hepatocyte growth factor followed by vascular endothelialgrowth factor, or (ii) hepatocyte growth factor followed bybasic-fibroblast growth factor from a bioerodable carrier platform. Inanother example, angiogenesis may be enhanced by sequential release of(i) vascular endothelial growth factor followed by platelet-derivedgrowth factor, or (ii) vascular endothelial growth factor followed byangiopoietin-I from a bioerodable carrier platform. In another example,cell recruitment followed by cell survival may be enhanced by sequentialrelease of (i) stromal derived factor-1-alpha followed by insulin-likegrowth factor-1, (ii) stem cell factor followed by insulin-like growthfactor 1, or (iii) granulocyte colony-stimulating factor followed byinsulin-like growth factor 1 from a bioerodable carrier platform. Inanother example, cell recruitment followed by angiogenesis may beenhanced by sequential release of (i) stromal derived factor-1-alphafollowed by vascular endothelial growth factor (ii) stem cell factorfollowed by vascular endothelial growth factor, (iii) stem cell factorfollowed by basic-fibroblast growth factor; or (iv) granulocytecolony-stimulating factor followed by basic-fibroblast growth factorfrom a bioerodable carrier platform. Any of the above examples may bereferred to as a binary sequential release formulation. In someembodiments, the binary sequential release formulation may include sonichedgehog protein. It should be appreciated that embodiments of theinvention encompass simultaneous or sequential release of more than twogrowth factors, or multiple simultaneous or sequential releaseformulation(s).

For sequential growth factor formulations, each growth factor may beencapsulated in a suitable combination of bioerodable carrier platforms,such as liposomes, polymerosomes, micelles, particles (e.g.,microparticles, nanoparticles, core-shell particles), nanofibers, orhydrogels. Alternatively, each growth factor may be associated with adifferent component of a single bioerodable carrier platform. In thecontext of this application, “component” means a different physical orchemical portion of the same platform. Sequential release formulationsmay be designed to release one growth factor at a faster rate thanrelease of a second growth factor. This may be achieved, for example,through use of different encapsulation (association) methods, such asliposomes for quick release and microparticles for longer, sustainedrelease. Additionally, adjusting polymer compositions (e.g., PEG for onegrowth factor and PLGA for a different growth factor) and/or molecularweights of the carrier platform(s) (e.g., in the case of polymer-basedcarriers) present other means with which to vary release and/ordegradation rates between different particles containing differentgrowth factors. In some embodiments, a first bioerodable carrierplatform is capable of release of a first growth factor in a timeinterval of between 10 seconds and 2 weeks and a second differentbioerodable carrier platform is capable of release of the seconddifferent growth factor in a time interval of between 10 seconds and 10weeks. For example, in an embodiment including microparticles andliposomes, the time interval for release of the liposome may be betweenabout 10 seconds and 24 hours, while the time interval for the releaseof the microparticle may be between about 10 seconds and 10 weeks. Inanother embodiment, a single bioerodable platform, such as anelectrosprayed core-shell particle, may be used for sequential release.

In some embodiments, a binary (or multiple) release formulation asdescribed above may include additional treatment agent(s). In oneexample, the formulation may include an inhibitor of matrixmetallopeptidase 9 (MMP-9). Matrix metalloproteinases are proteolyticenzymes which are believed to be involved in the process of cardiacremodeling following an MI. Early experimental studies have shown that amatrix metalloproteinase inhibitor is effective in reversing the leftventricle remodeling process which may ultimately lead to heart failure.Therefore, it is anticipated that addition of an inhibitor of MMP-9 tobinary (or multiple) release formulations will have an increasedbeneficial effect on the injury site. Examples of MMP-9 inhibitorsinclude, but are not limited to, tissue inhibitor of metalloproteinase-1(TIMP-1), TIMP-3, TIMP-4, nobiletin, indole-3-carbinol, batimastate,marimastat, solimastat, neovastat, Bay 12-9566, AG3340, COL-3,BMS-275291, or CGS27023A.

In some embodiments, a binary (or multiple) release formulation asdescribed above may include a specific IGF-1 isoform such as majorextrahepatic insulin-like growth factor-1 (mIGF-1). mIGF-1 is naturallyabundant in skeletal muscles and is believed to be more effective forcardiac muscle regeneration relative to other IGF-1 isoforms. At leastone study has shown that mIGF-1 has the ability to modulate theinflammatory response with minimum scar formation. See Musaro, A., etal., The Role of local Insulin-like Growth Factor-1 Isoforms in thePathophysiology of Skeletal Muscle, Current Genomics, vol. 3, pp.149-162 (2002).

In some embodiments, a binary (or multiple) release formulation asdescribed above may include a cardiovascular treatment agent useful inthe treatment of compromised cardiac tissue. Examples of suchcardiovascular treatment agent include, but are not limited to,antiplatelet drugs (e.g., aspirin, clopidogrel, ticlopidine, cilostazol,abciximab, eptifibatide, tirofiban, dipyridamole); beta blockers (e.g.,aprenolol, carteolol, levobunolol, mepindolol, metapranolol, nadolol,oxprenolol, penbutolol, pindolol, propranolol, sotalol, timolol,acebutalol, atenolol, betaxolol, bisoprolol, esmolol, metprolol,nebivolol, carvedilol, celiprolol, labetalol, butaxamine);angiotensin-converting enzyme (ACE) inhibitors (e.g., captopril,enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril,fosinopril, casokinins, lactokinins); statins (e.g., atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin, simvastatin, simvastatin/ezetimibecombination, lovastatin/niacin extended release combination, i.e.,ADVICOR by Abbott Laboratories, Illinois, U.S.A.,atorvastatin/amlopidine besylate, mesylate, or maleate combination),omega-3 fatty acids (natural or synthetic); or aldosterone antagonists(e.g., eplerenone).

Methods of Treatment

Techniques for delivering a binary (or multiple) release formulationaccording to embodiments of the invention to an injury site include, butare not limited to, percutaneous delivery, such as intra-coronarydelivery, intra-myocardial delivery, and periadventitial delivery, inaddition to delivery through an open chest procedure. Intra-coronarydelivery includes antegrade arterial delivery, which typically involvesrouting a catheter through the arterial vasculature, and retrogradevenous delivery, which typically involves routing a catheter through thevenous vasculature. In intra-coronary delivery, the binary (or multiple)release formulation is delivered in the vasculature system adjacent theinfarct region and its respective border zone. In intra-myocardialdelivery, the binary (or multiple) release formulation is delivereddirectly into the ventricular wall by a needle. Periadventitial deliveryrefers to delivery of drugs from across the adventitia of arteries aswell as delivery in the pericardial sac.

FIGS. 1A-1C illustrate an embodiment of a needle injection device whichmay be used to deliver a binary (or multiple) release formulation inaccordance with embodiments of the invention. In general, the catheterassembly 100 provides a system for delivering substances to or through adesired area of a blood vessel or organ, such as a coronary artery orthe heart, in order to treat a post-MI region or ischemic HF region. Thecatheter assembly 100 is similar to the catheter assembly described incommonly-owned, U.S. Pat. Nos. 7,179,249 and 6,554,801, titled“Directional Needle Injection Drug Delivery Device and Method of Use,”by Steward, J. et al. which are incorporated herein by reference.

In one embodiment, catheter assembly 100 is defined by elongatedcatheter body 150 having proximal portion 120 and distal portion 110.Guidewire cannula 170 is formed within catheter body (from proximalportion 110 to distal portion 120) for allowing catheter assembly 100 tobe fed and maneuvered over guidewire 180. Balloon 130 is incorporated atdistal portion 110 of catheter assembly 100 and is in fluidcommunication with inflation cannula 160 of catheter assembly 100.

Balloon 130 may be formed from balloon wall or membrane 135 which isselectively inflatable to dilate from a collapsed configuration to adesired and controlled expanded configuration. Balloon 130 may beselectively dilated (inflated) by supplying a fluid into inflationcannula 160 at a predetermined rate of pressure through inflation port165 (located at proximal end 120). Balloon wall 135 is selectivelydeflatable, after inflation, to return to the collapsed configuration ora deflated profile. Balloon 130 may be dilated (inflated) by theintroduction of a liquid into inflation cannula 160. Liquids containingtreatment and/or diagnostic agents may also be used to inflate balloon130. In one embodiment, balloon 130 may be made of a material that ispermeable to such treatment and/or diagnostic liquids. To inflateballoon 130, the fluid may be supplied into inflation cannula 160 at apredetermined pressure, for example, between about one and 20atmospheres. The specific pressure depends on various factors, such asthe thickness of balloon wall 135, the material from which balloon wall135 is made, the type of substance employed and the flow-rate that isdesired. In some embodiments, a balloon may be necessary to temporarilyocclude a blood vessel so that the natural flow of blood does notinterrupt the procedure by preventing proper placement of the injectionneedle. In other embodiments, a balloon may be necessary to localize theinjection needle near the target region, or compromised heart tissue.

In this embodiment, catheter assembly 100 also includes at least twosubstance delivery assemblies 105 a and 105 b (not shown; see FIGS.1B-1C) for injecting a binary (or multiple) release formulation to amyocardial infarct region or other treatment region. In one embodiment,substance delivery assembly 105 a includes needle 115 a movably disposedwithin hollow delivery lumen 125 a. Delivery assembly 105 b includesneedle 115 b movably disposed within hollow delivery lumen 125 b (notshown; see FIGS. 1B-1C). Delivery lumen 125 a and delivery lumen 125 beach extend between distal portion 110 and proximal portion 120.Delivery lumen 125 a and delivery lumen 125 b may be made from anysuitable material, such as polymers and copolymers of polyamides,polyolefins, polyurethanes and the like. Access to the proximal end ofdelivery lumen 125 a or delivery lumen 125 b for insertion of needle 115a or 115 b, respectively is provided through hub 135 (located atproximal end 120). Delivery lumens 125 a and 125 b may be used todeliver a binary (or multiple) release formulation to a post-myocardialinfarct region. In some embodiments, it may be helpful to maximizedistribution of the formulation by using a dual needle catheter. Itshould be appreciated, however, that a single needle catheter and anon-needle catheter may be appropriate for delivery of a binary (ormultiple) release formulation. Intra-coronary delivery methods typicallyutilize non-needle-based catheters while intra-myocardial deliverymethods typically utilize needle-based catheters.

FIG. 1B shows a cross-section of catheter assembly 100 through line A-A′of FIG. 1A (at distal portion 110). FIG. 1C shows a cross-section ofcatheter assembly 100 through line B-B′ of FIG. 1A. In some embodiments,delivery assemblies 105 a and 105 b are adjacent to each other. In thecase where the biodegradable carrier platform is a two-componenthydrogel, the proximity of delivery assemblies 105 a and 105 b allowseach component of the hydrogel to rapidly gel when delivered to atreatment site, such as a post-myocardial infarct region.

FIG. 2 illustrates an embodiment of a syringe which may be used pursuantto embodiments of the invention. Syringe 200 may include a body 205, aneedle 210 and a plunger 215. A shaft of plunger 215 has an exteriordiameter slightly less than an interior diameter of body 205 so thatplunger 215 may, in one position, retain a substance in body 205 and, inanother position, push a substance through needle 210. Syringes areknown by those skilled in the art. In some applications, syringe 200 maybe applied directly to a treatment site during an open-chest surgeryprocedure to deliver core-shell particles to a treatment site.

From the foregoing detailed description, it will be evident that thereare a number of changes, adaptations and modifications of the presentinvention which come within the province of those skilled in the part.The scope of the invention includes any combination of the elements fromthe different species and embodiments disclosed herein, as well assubassemblies, assemblies and methods thereof. However, it is intendedthat all such variations not departing from the spirit of the inventionbe considered as within the scope thereof.

What is claimed is:
 1. A composition, comprising: a first bioerodablecarrier platform capable of sustained release of a first growth factor;at least one second different bioerodable carrier platform capable ofsustained release of a second growth factor, wherein the seconddifferent bioerodable carrier platform comprises at least two precursorsof a two-component hydrogel, wherein the at least two precursors areoperable to have a first liquid state in which they are capable ofseparate delivery and a second semi-solid gel state which forms when theat least two precursors mix; and at least one cell type, wherein a rateof release of the first growth factor from the first bioerodable carrierplatform is faster than a rate of release of the second growth factorfrom the second bioerodable carrier platform, and wherein the firstgrowth factor and the second growth factor are different and each of thefirst growth factor and the second growth factor have a differentfunction when delivered to the compromised cardiac tissue such that cellsurvival followed by cell homing is enhanced.
 2. The composition ofclaim 1 wherein the two-component hydrogel is selected from the groupconsisting of hyaluronic acid, fibrin glue, alginate and silk elastincopolymers.
 3. The composition of claim 1 wherein the at least one celltype selected from the group consisting of localized cardiac progenitorcells, cardiac stem cells, mesenchymal stem cells, bone marrow derivedmononuclear cells, adipose-derived stem cells, embryonic stem cells,umbilical cord blood derived stem cells, smooth muscle cells, skeletalmyoblasts, endothelial progenitor cells and a combination thereof. 4.The composition of claim 1 wherein one of the first or second carrierplatforms is one of a liposome, a polymersome, a micelle, a particle, acore-shell particle, a hydrogel, a fiber, or a microbubble, wherein whenthe carrier platform is a polymersome, a particle, or a hydrogel, thecarrier platform is fabricated from a material selected from the groupconsisting of fibrin glue, alginate gel, small intestinalsubmucosa/urinary bladder matrix, poly(ethylene glycol)-based materials,poly(N-isopropylacrylamide) copolymers, poly(D,L-lactide-co-glycolide),polyurethanes, polyurethane-ureas, poly(ester) copolymers, dextran,silk-elastin, hyaluronate, or a combination or a precursor thereof. 5.The composition of claim 1 wherein the first growth factor and thesecond growth factor comprise one of: (i) insulin-like growth factor 1and hepatocyte growth factor; (ii) insulin-like growth factor 1 andstromal derived factor-1-alpha; (iii) vascular endothelial growth factorand platelet-derived growth factor; (iv) vascular endothelial growthfactor and hepatocyte growth factor; (v) vascular endothelial growthfactor and stem cell factor; (vi) vascular endothelial growth factor andstromal derived factor-1-alpha (vii) basic-fibroblast growth factor andhepatocyte growth factor; (viii) basic-fibroblast growth factor and stemcell factor; (ix) insulin-like growth factor 1 and basic-fibroblastgrowth factor; (x) granulocyte colony-stimulating factor and hepatocytegrowth factor; (xi) granulocyte colony-stimulating factor and hepatocytegrowth factor; (xii) granulocyte colony-stimulating factor and stem cellfactor; (xiii) granulocyte colony-stimulating factor and insulin-likegrowth factor 1, or (xiv) granulocyte colony-stimulating factor andstromal derived factor 1-alpha, the composition optionally includingsonic hedgehog protein.
 6. The composition of claim 1 wherein the firstgrowth factor and the second growth factor comprise one of: (i)insulin-like growth factor 1 and stromal derived factor-1-alpha; (ii)insulin-like growth factor 1 and hepatocyte growth factor; (iii)insulin-like growth factor-1 and stem cell factor, (iv) insulin-likegrowth factor 1 and granulocyte colony-stimulating factor; (v)hepatocyte growth factor and vascular endothelial growth factor; (vi)hepatocyte growth factor and beta-fibroblast growth factor; (vii)vascular endothelial growth factor and platelet-derived growth factor;(viii) vascular endothelial growth factor and angiopoietin-I; (ix) stemcell factor and insulin-like growth factor 1; (x) granulocytecolony-stimulating factor and insulin-like growth factor 1; (xi) stemcell factor and vasoendothelial growth factor; (xii) stem cell factorand beta-fibroblast growth factor; or (xiii) granulocytecolony-stimulating factor and beta-fibroblast growth factor, thecomposition optionally including sonic hedgehog protein.
 7. Thecomposition of claim 5 or 6, further comprising, at least one of: (i) amatrix metallopeptidase 9 (MMP-9) inhibitor comprising one of tissueinhibitor of metalloproteinase-1 (TIMP-1), TIMP-3, TIMP-4, nobiletin,indole-3-carbinol, batimastat, marimastat, solimastat, neovastat, Bay12-9566, AG3340, COL-3, BMS-275291, or CGS27023A; (ii) majorextrahepatic insulin-like growth factor-1; or, (iii) a cardiovasculartreatment agent selected from the group consisting of an antiplateletdrug, a beta blocker, an angiotensin-converting enzyme inhibitor, astatin, an omega-3 fatty acid, or an aldosterone antagonist.
 8. Thecomposition of claim 1 wherein the first bioerodable carrier platform iscapable of simultaneous release of the first treatment agent and atleast one other treatment agent in a time interval of between 10 secondsand 10 weeks.
 9. The composition of claim 1 wherein the firstbioerodable carrier platform is capable of release of the firsttreatment agent in a time interval of between 10 seconds and 10 weeksand the second different bioerodable carrier platform is capable ofrelease of the second treatment agent in a time interval sequential tothat of the first treatment agent.
 10. The composition of claim 1wherein the first bioerodable carrier platform is capable of release ofthe first treatment agent in a time interval of between 10 seconds and24 hours and the second different bioerodable carrier platform iscapable of release of the second treatment agent in a time intervalsequential to that of the first treatment agent.
 11. The composition ofclaim 1 wherein the first bioerodable carrier platform includesarginine-aspartate-glycine (RGD).
 12. The composition of claim 1 whereinthe first bioerodable carrier platform is a liposome and the seconddifferent bioerodable carrier platform is a hydrogel.
 13. Thecomposition of claim 1 wherein the first growth factor comprisesinsulin-like growth factor
 1. 14. The composition of claim 13 whereinthe second growth factor comprises stromal derived factor-1-alpha. 15.The composition of claim 1 wherein the at least one cell type comprisescardiac progenitor cells.
 16. A composition, comprising: a firstbioerodable carrier platform capable of sustained release of a firsttreatment agent and a second treatment agent simultaneously, wherein thesecond treatment agent is different from the first treatment agent; atleast one second different bioerodable carrier platform capable ofsustained release of a third treatment agent wherein a rate of releaseof the first bioerodable carrier platform is different relative to arate of release of the second bioerodable carrier platform such that thethird treatment agent is delivered sequentially to the first treatmentagent and the second treatment agent, and wherein the second differentbiodegradable carrier platform comprises at least two precursors of atwo-component hydrogel, wherein the at least two precursors are operableto have a first liquid state in which they are capable of separatedelivery and a second semi-solid gel state which forms when the at leasttwo precursors mix, wherein the first treatment agent and the secondtreatment agent are different from the third treatment agent and atleast two of the first treatment agent, the second treatment agent andthe third treatment agent have a different function when delivered tocompromised cardiac tissue, the function selected from the groupconsisting of cell survival, cell recruitment, angiogenesis,arteriogenesis, and anti-fibrotic development.
 17. The composition ofclaim 16 wherein the function of the third treatment agent is dependenton the function of one of the first treatment agent and the secondtreatment agent.
 18. The composition of claim 16 wherein the firsttreatment agent and the second treatment agent, when releasedsimultaneously, function to enhance cell survival at the compromisedcardiac tissue and sequential release of the third treatment agentenhances the function of the first and second treatment agents.
 19. Thecomposition of claim 16 wherein the first treatment agent isinsulin-like growth factor 1, the second treatment agent is hepatocytegrowth factor and the third treatment agent is stromal derivedfactor-1-alpha such that cell survival at the compromised cardiac tissueis enhanced followed by enhanced cell homing.